The ARF tumor suppressor protein is encoded by the Ink4a/ARF gene, which is the second most frequently mutated genetic locus in human cancer. To date the majority of research on ARF has focused on its tumor suppressor functions. This proposal focuses instead on a novel survival function for ARF that we have recently uncovered, and that exists for a subset of tumors with mutant/null p53. We show that ARF plays an integral role in starvation-induced autophagy. Autophagy is a self-catabolic process that promotes the survival of cells exposed to nutrient deprivation. Because tumor cells exist under uniquely metabolically-stressed conditions, many rely heavily on this pathway in order to subsist. Consistent with this fact, autophagy inhibitors have shown promise as anti-cancer agents. We show that ARF protein is markedly up-regulated in response to nutrient deprivation. We show that silencing ARF impedes autophagy and decreases the survival of nutrient-deprived cells. Finally, we show that silencing ARF in lymphomas impairs autophagy and survival, and actually impedes the development of these tumors. The central hypothesis of this proposal is that ARF-mediated autophagy is utilized by a subset of tumors with mutant p53 to allow them to survive episodes of metabolic stress. We contend that the requirement for the survival function of autophagy is an 'Achilles heel'for tumors. We need to better understand this pathway in order to exploit it for cancer therapy. In proposed research we will elucidate the mechanism(s) whereby ARF induces autophagy. We will determine how nutrient deprivation leads to increased ARF protein levels. We have found that ARF expression is beneficial for some tumors (lymphoma) but not others (sarcoma);we will define the subset of tumors that are benefited by ARF-mediated autophagy. We will determine whether critical mediators of autophagy, such as ARF and Beclin1, dictate tumor response to small molecules that modulate this process, such as chloroquine (inhibits autophagy), and trehalose (induces autophagy). Finally we will elucidate the mechanism of transcriptional repression of the ARF locus by p53, as this leads to ARF up-regulation in p53-mutant tumors. The combined data will serve as a necessary foundation for exploiting the pathway of autophagy for cancer therapy. PUBLIC HEALTH RELEVANCE: The ARF gene is encoded by the Ink4a/ARF locus, which is the second most commonly mutated genetic locus in human cancer;up to 50% of human tumors contain mutations in Ink4a/ARF. This fact argues that research aimed at understanding the basic function of ARF in human cancer is warranted. We show that ARF controls a key survival pathway for tumor cells. We believe that the requirement for ARF and autophagy may be an Achilles Heel for tumor cells. This research is aimed at understanding this pathway, and at finding ways to manipulate this pathway to combat cancer.